Combination of reader and incubator

ABSTRACT

The present invention provides a detection arrangement for detecting presence of an analyte in a sample, comprising a processor, a memory, a display, and a color measuring device, characterized in that a means is present for maintaining a constant temperature or a temperature profile of said sample. Furthermore, the present invention provides a method for determining the presence or absence of an analyte in a fluid by analysis of image data from an assay that generates an image result on an assay medium, comprising the steps of: (a) incubating a sample of said fluid together with said assay at a pre-set temperature or temperature profile (b) obtaining said image result on an assay medium; and (c) imaging the image result with an image acquisition device to generate digital image data corresponding to the image result; and (d) using data processing means, applying to the digital image data a stored relationship between the image result and assay calibration data to generate a quantified result for said assay, characterized in that incubation step (a) is carried out simultaneously with steps (b)-(c).

FIELD OF THE INVENTION

The present invention relates to an improved novel assay system and anew method for the rapid determination of the presence or absence ofanalytes in a sample using simple reading- and computer equipmentcombined with heating equipment.

BACKGROUND OF THE INVENTION

Test methods (assays) for the detection of analytes in samples arewidely known in the art. Particular examples of assays are those wherebythe analyte to be detected has a specific color-, IR-, or UV-spectrumand can thus be detected by means of a reader comprising a photoelectriccell, a scanner, any type of camera and of course visually. If thespectral characteristics of the analyte in question are not sufficientfor adequate detection it is often possible to use indirect methods. Forinstance, methods are available whereby the presence of the analytetriggers a secondary process, which then results in the formation of aproduct or event having a specific spectral characteristic. This may bea colored product, but can also be a change in pH, redox-potential,temperature and the like, which in turn triggers an indicator moleculeto change color-, IR-, redox- or UV-spectrum. Many types of secondaryprocesses are employed in the art for various applications.

One example is a chemical reaction of the analyte with a fluorescent orcolored molecule resulting in a product without fluorescence or color orwith a different fluorescence or color, or the other way around.

Another example is inhibition of a (bio) chemical process by the analytewhereby the presence of the analyte is indirectly measured as a resultof the absence (or presence) of the effect of that (bio) chemicalprocess. The latter may be illustrated by, for instance, microbiologicalassays for the detection of analytes, particularly residues ofantibiotics and chemotherapeutics, in fluids such as milk, meat juice,serum, urine, (waste) water and the like. Examples of such assays havebeen described in CA 2056581, DE 3613794, EP 0005891, EP 0285792, EP0611001, GBA 1467439 and U.S. Pat. No. 4,946,777. These descriptions alldeal with ready to use assays that make use of an assay organism andwill give a result by the change indicated by an indicator molecule, forinstance a change of color of a pH- and/or redox-indicator, added to theassay. A change in the indicator indicates the presence of a growingassay organism. The principle is that when an analyte is present in afluid in a concentration sufficient to inhibit growth of the assayorganism, the color of the indicator will stay the same. In contrast,when no inhibition occurs, growth of the assay organism is accompaniedby the formation of acid or reduced metabolites or other phenomena thatwill induce an indicator signal. The known assays mentioned aboveinclude an assay medium, such as an agar medium, inoculated with asuitable assay organism, preferably a strain of Bacillus, Escherichia orStreptococcus, and a pH indicator and/or a redox indicator. The suitableassay organism and the indicator, and optional buffers, nutrients,surfactants and the like, are introduced into a gel or simply kept insolution. Normally conditions are chosen in such a way that the assayorganisms stay alive but cannot multiply because of lack of an essentialgrowth requirement (this may be a nutrient, a specific pH- ortemperature value or any other essential parameter).

Assays that require a more or less stringent temperature regime for theresults to be generated reliably and accurately form a special class ofmethods amongst the ones mentioned above. These types of methods requireincubation equipment in order to maintain the assay at a predeterminedtemperature or temperature profile for a given period of time. Usuallythe result of such an assay is determined afterwards by using a readerequipped to detect the event that indicates the presence of the analyte.An example of such an assay is described in WO 03/033728 dealing with amethod for detecting the presence of an analyte (such as a β-lactam) ina sample (such as food products, e.g. meat, milk, or body fluids, e.g.blood, urine) following incubation at 64° C. by determining color valuesof the sample, associated with the L*a*b color model, using a standardscanner coupled to a computer. The latter reading technology was alsodisclosed in EP 953 149.

A severe disadvantage of these prior art methods is that it is onlypossible to perform reading operations after incubation. As a result ofthis, diagnostic methods such as microbiological assays for thedetection of antimicrobial residues only give a positive or negativeresult (i.e. merely indicate whether or not the concentration of analyteis above or below a certain threshold value). In case of a positiveresult obtained with these so-called screening assays, the sample has tobe examined further for confirmation using a second diagnostic method.Mostly, such confirmation methods, e.g. HPLC or mass spectrometry, areextremely expensive and it takes a long time before the results areknown. Thus, reading assay results during the screening assay itselfwould be advantageous, as this would give access to more detailedinformation that could improve on parameters such as assay duration,type of analyte or concentration of analyte.

Nevertheless, reading of information during an assay and simultaneousincubation of such an assay is known in methods based on the presence ofa light source on one end of the assay and collecting light signals onthe other end of the assay, an example of which are the well-knownELISA-readers. This principle has, unfortunately, three majordisadvantages. Firstly, in assays wherein the sample is placed on asubstrate and the required reaction takes place within the substrate,the nature and amount of the sample will disturb the measurement whenlight or another type of radiation travels through both sample andsubstrate. Secondly, the equipment usually employed for suchmeasurements is mostly dedicated and designed for trained personnel andto be used in a laboratory environment. Thirdly, the prior artprinciples are designed to measure one particular wavelength only. Thereis thus a need for equipment and methods that do not suffer from thesedrawbacks.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a detectionarrangement for detecting presence of an analyte in a sample, comprisinga processor, a memory, a display, and a color measuring device, saidmemory, said display and said color measuring device being arranged tocommunicate with said processor, said color measuring device beingarranged to generate light signals, to send said light signals to saidsample, to receive return light signals from said sample, to convertsaid return light signals into color signals and to send said colorsignals to said processor, said processor being operated by instructionsstored in said memory and being arranged to calculate a value of acomposite parameter Z in accordance with a following equation:

$Z = {\sum\limits_{i = 1}^{n}\; {w_{i}x_{i}}}$

where x_(i) is a color signal i and w_(i) is a corresponding weighingfactor and i is an index ranging from 1 to n and n is an integer equalto the number of color signals, characterized in that a means is presentfor maintaining a constant temperature or a temperature profile of saidsample.

Furthermore, it is an object of the present invention to provide amethod for determining the presence or absence of an analyte in a fluidby analysis of image data from an assay that generates an image resulton an assay medium, comprising the steps of:

-   -   (a) incubating a sample of said fluid together with said assay        at a pre-set temperature or temperature profile    -   (b) obtaining said image result on an assay medium; and    -   (c) imaging the image result with an image acquisition device to        generate digital image data corresponding to the image result;        and    -   (d) using data processing means, applying to the digital image        data a stored relationship between the image result and assay        calibration data to generate a quantified result for said assay,        characterized in that incubation step (a) is carried out        simultaneously with steps (b)-(c).

DETAILED DESCRIPTION OF THE INVENTION

The terms and abbreviations given below are used throughout thisdisclosure and are defined as follows.

The term ‘assay medium’ refers to a composition such as a solution, asolid or, preferably, in the form of a sol or a gel, for instancecomprising a gelling agent. Suitable examples of gelling agents areagar, alginic acid and salts thereof, carrageenan, gelatin,hydroxypropylguar and derivatives thereof, locust bean gum (Carob gum),processed eucheuma seaweed and the like. However, the person skilled inthe art will understand that other types of solid assay media may bebased on carrier materials such as ceramics, cotton, glass, metalparticles, paper, and polymers in any shape or form, silicates, sponges,wool and the like. Usually, an assay medium contains one or moreindicators; however, these compounds may also be added later when theassay is being performed. The assay medium may comprise one or moretypes of assay organisms or enzymes as detecting agents and nutrients.Optionally, the assay medium may also contain one or more buffers,stabilizers, substances that change the sensitivity to certain analytesin a positive or negative way, and/or viscosity-increasing agents.Examples of substances that change the sensitivity to certain analytesare antifolates like ormethoprim, tetroxoprim and trimethoprim thatimprove the sensitivity of the assay organism towards sulfa compounds orsalts of oxalic acid or hydrofluoric acid, which improve the sensitivitytowards tetracycline. Examples of viscosity-increasing agents areascorbyl methylsilanol pectinate, carbomer, carboxymethyl cellulose,cetearyl alcohol, cetyl alcohol, cetyl esters, cocamide DEA, emulsifyingwax, glucose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose,lauramide DEA, linoleamide DEA, magnesium aluminum silicate,maltodextrins, PEG-8 distearate, polyacrylamide, polyvinyl alcohol,PVP/hexadecene copolymer, sodium chloride, sodium sulfate,soyamidopropyl betaine, xanthan gum and the like. The assay medium maybe contained within any type of container; frequently used containersare tubes, microtiter plates and Petri dishes.

The term ‘CFU’ is an abbreviation of Colony Forming Units and refers tothe number of microorganisms, spores of microorganisms, partiallygerminated spores of microorganisms or vegetative cells capable ofproducing colonies of microorganisms.

The term ‘reader’ refers to a device capable of capturing color or otherspectral images and translating these images into analogous or digitalsignals, such as a scanner, a digital photo camera or video camera, aweb cam apparatus or the like.

The term ‘fluid’ refers to a substance (as a liquid, not a gas) tendingto flow or conform to the outline of its container.

The term ‘gelling agent’ refers to a compound that assists in changing amixture into or taking on the form of a gel.

The term ‘indicator’ refers to a substance used to measure (for exampleby change of color or fluorescence) the condition of an assay mediumwith respect to the presence of a particular material (for example anacid, a base, oxidizing or reducing agents). For instance, the term‘indicator’ may refer to one or more compounds that are known aspH-indicators, but also to one or more compounds that are known asredox-indicators. Also, the term ‘indicator’ may refer to mixtures oftwo or more different types of indicators, such as a combination of apH- and a redox-indicator. In general, when two or more indicators areused, these indicators are co-operating to increase the indicator effectof each of the indicators when taken alone.

The term ‘sensitivity’ refers to the degree of receptiveness of a givensystem to sense a certain state. More particularly, in the present case‘sensitivity’ refers to the degree by which concentrations of analytesin a sample can be determined.

The term ‘spore’ refers to a primitive usually unicellular oftenenvironmentally resistant dormant or reproductive body produced bymicroorganisms and capable of development into a new individualmicroorganism.

The term ‘temperature profile’ refers to a range of temperature valuesas a function of time. This may be a linear range, a hyperbolic range,an exponential range, a polynomial range but also any range suitable fora specific application and not necessarily described by a simplemathematical function. A pre-set temperature is a species of the genustemperature profile as it is one temperature value maintained the samethroughout time.

The term ‘threshold’ refers to the concentration value above which agiven analyte is to be regarded as present (positive) and below whichsaid analyte is to be regarded as absent (negative). Generally, athreshold value is given for particular analytes in particular samplesby local, regional or interregional authorities but it can also bepre-set for certain research purposes. Alternatively, a threshold valuemay be a derivative of a formula, such as a slope.

The term ‘transparent’ refers to any material that allows for light totravel through the material. Ideally a transparent material does notretain any proportion of the light as it is send in, however since suchmaterial is not known, the term ‘transparent’ particularly refers tomaterials that allow for light to travel through with minimal losses.Minimal losses may be in the range below 50%.mm⁻¹, preferably below20%.mm⁻¹, more preferably below 10%.mm⁻¹, still more preferably below5%.mm⁻¹, most preferably below 1%.mm⁻¹.

In a first aspect of the present invention there is provided a detectionarrangement for detecting presence and concentration of an analyte in asample, comprising a processor, a memory, a display, a communicationdevice such as a corn port or USB port and a spectrum measuring device,said memory, said display and said measuring device being arranged tocommunicate with said processor and arranged to generate light signals,to send said light signals to said sample, to receive light signals fromsaid sample, to convert said light signals into color signals and tosend these color signals to the processor whereby a means is present formaintaining a constant temperature or temperature profile of saidsample. Such a means may be an incubator comprising a heating device andan aperture for holding a sample. Either the measuring device is locatedwithin the heating device or the heating device is located within themeasuring device. Although the detection arrangement may comprisegeneration of light signals at one source followed by detection of lightsignal at a source opposite the first source, in a preferred embodiment,the detection arrangement avoids the problems associated with thetraveling of light through sample and substrate as it is based on theprinciple of reflection of light. Hence, using reflection of light, itis for instance no longer required to remove the sample from thesubstrate prior to irradiation, which in turn has the advantage that abroader part of the fight spectrum (i.e. also including infrared) can beused. Moreover, the above can be realized using easily accessible andrelatively cheap equipment such as a digital camera, a scanner, aweb-cam or the like.

In a first embodiment of the invention, said means for maintaining aconstant temperature or temperature profile of a sample and an assaymedium is a device comprising one or more apertures for holding assaymedium and/or samples. Samples and/or assay medium may be introduceddirectly into the apertures but alternatively can also be present incontainers of which at least the bottom is transparent. The device has atransparent bottom side, at least at those places where sample and/orassay medium are located. Temperature maintenance is realized byconstructing at least part of the device from a material that warms upwhen an electrical current is applied, or alternatively by constructingat least part of the device from a material through which a fluid with apre-set temperature can be circulated. As an example, said means formaintaining a constant temperature or temperature profile of the sampleand/or assay is a transparent plate, for instance made of glass or anykind of plastic. The transparent plate is coated with a material thatcan be heated, for instance by applying an electrical current.Advantageously, said material is transparent or semi-transparent. In thelatter case the material will allow light to travel through when appliedin a sufficiently thin layer, preferably having a thickness ranging from0.01-200 μm, more preferably ranging from 0.1-50 μm, most preferablyranging from 1-20 μm, still most preferably ranging from 5-15 μm.Titanium dioxide is a material that is particularly suitable forobtaining the desired effect, but also other materials known in the artmay be used. The size of the particles preferably ranges from 0.1-100nm, more preferably from 1-50 nm, most preferably from 10-30 nm. Inorder to heat the material and hence the plate supporting the samples tobe analyzed, a means for applying an electrical current is attached tothe plate. Such a means may be a set of electrodes attached to the plateand contacted with the material. The electrodes can be made of anymaterial that allows for transport of electrical current; attachment ofthe electrodes can be effected with glue capable of transportingelectrical current. Such electrodes and glues are well known to theperson skilled in the art. Optionally, the temperature is measured bymeans of a temperature sensor similarly attached to the plate andconnected to the processor. Optionally also the electrodes are connectedto the processor and the processor is instructed to keep the temperatureof the plate at a pre-set value or profile by applying the requiredcurrent to the electrodes. Samples and/or assay medium may be introduceddirectly into the apertures of the heating device but alternatively canalso be present in containers of which at least the bottom istransparent. The device has a transparent bottom side, at least at thoseplaces where sample and/or assay medium are located. Temperaturemaintenance is realized by constructing at least part of the device froma material that warms up when an electrical current is applied.

In another embodiment of the invention, said means for maintaining aconstant temperature or temperature profile of a sample and an assaymedium is a device comprising one or more apertures for holding assaymedium and/or samples. Attached to said device are heat traces, forinstance made from metals such as aluminum, copper, gold, lead, silver,tin and the like that allow for generating a specific heat input forhomogeneous temperature gradients. To increase accuracy of thetemperature gradient over the device, one or more temperature sensorsand/or controllers are optionally included to adjust the current input.In an example, said temperature sensors are electrically integrated in aprint plate and are in thermal contact with said device which is made ofa heat conducting material, such as a metal like aluminum, copper oriron. The temperature is controlled by means of a Proportional IntegralDerivative (PID) controller or any other type of controller orcombination thereof. To increase speed of detection and reproducibilitythe heating device may be pre-heated at a higher temperature.

In the Figures and the legends thereto an example is given of thedetection arrangement (FIG. 1) and the heating device (FIG. 2) of thepresent invention. Although these Figures are not intended as limitingthe scope of the invention, they will allow the person skilled in theart to reproduce the invention.

In a second aspect of the invention there is provided a method fordetermining the presence or absence of an analyte in a fluid by analysisof image data from an assay that generates an image result on an assaymedium. More specifically, said assay, together with a sample of thefluid to be analyzed, requires to be incubated at a pre-set temperatureor temperature profile. Temperatures or temperature profiles depend onthe nature of the assay. For instance, in microbiological inhibitionassays, microorganisms usually require a temperature ranging from 25-45°C., preferably from 30-40° C., more preferably from 35-39° C. However,other microorganisms such as thermophilic microorganisms (i.e. Bacillusstearothermophilus and others) require quite different temperatures foroptimal growth, i.e. ranging from 40-75° C., preferably from 50-70° C.,more preferably from 60-68° C. and most preferably from 63-66° C.Advantageously, the method of the present invention provides forsimultaneous incubation of the assay, obtaining an image result on anassay medium and imaging of the image result with an image acquisitiondevice to generate analog and/or digital image data corresponding to theimage result. Also simultaneously, a data processing means may be usedto apply to the digital image data, a stored relationship between theimage result and assay calibration data, such as a standard color card,in order to generate a quantified result for said assay. Alternatively,multiple measurements based on color differences in time may be used toobtain said quantified result. The latter alternative has the advantagethat calibration may be circumvented.

In a first embodiment of the second aspect of the invention, a pre-settemperature or temperature profile is maintained by means formaintaining a constant temperature or temperature profile as outlined inthe first aspect of the invention. Advantageously said means is heatedat the same temperature at every point of said means. Preferably, thedifferences in temperature between individual points of said means arenot more than 10° C., preferably not more than 5° C., more preferablynot more than 2° C., and most preferably not more than 1° C.

In a second embodiment image results are obtained continuously or atregular intervals (i.e. at least twice and the time interval between twoconsecutive measurements is 0.00001 to 200 minutes) or at irregularintervals in order to establish any changes as early as possible.Preferably, said time interval is between 0.1 min and 160 min, morepreferably between 0.5 min and 120 min, and most preferably between 1min and 10 min. Measuring color values as a function of time offersseveral substantial advantages.

In the first place, many of the assays used in the art suffer thedisadvantage of reducing durability. As a result of this, fixed assayduration inevitably leads to a lower accuracy. The reason for this isthat, the older a given assay is, the longer it will take to obtain acertain result. Establishing the required test duration by means ofrunning a blank sample may circumvent this, although this is acumbersome methodology which still lacks optimal accuracy. By obtainingresults as a function of time however, this drawback is now circumventedas the exact point in time where a given color parameter changes can nowbe measured precisely during the assay. Consequently, by using themethod of the present invention, the assay storage life, although stillan important parameter for the suitability of an assay per se, is nolonger decisive for the accuracy of the assay.

In the second place, by taking measurements early during an assay, thesensitivity of the assay increases. For instance, in microbiologicalinhibition assays, growth inhibition of microorganisms occurs at loweranalyte concentrations than later on during the assay when theinhibitory effect of the analytes begins to diminish. Advantageously,depending on the type of analyte, the results of microbial inhibitionassays can be obtained in hitherto unprecedented short times, i.e.within 120 minutes, within 90 minutes or even within 60 or 30 minutes.

In the third place, rather than increasing the sensitivity by measuringearly during an assay, the method of the present invention allows forfurther speeding up analysis times in microbial inhibition assays byincreasing the amount of microorganisms. Whereas in prior art methodsthis approach would lead to decreasing sensitivity, measuring as afunction of time as in the present invention makes it possible tocounteract the decrease in sensitivity by shortening the assay duration.As an example, prior art microbial inhibition assays usually employmicroorganism concentrations ranging from 10⁴-10⁹ CFU.ml⁻¹, whereas inthe present invention these concentrations can be increased without lossof sensitivity two-fold to even 1000-fold. Hence, suitable and fastassays can be achieved using microorganism concentrations up to 10⁹,5×10⁹, 10¹⁰, 5×10¹⁰, 10¹¹ or even 10¹² CFU.ml⁻¹, giving assay durationsranging from 5-120 min, preferably 30-100 min, more preferably 45-90min.

In the fourth place, the method of the present invention allows for theeasy incorporation of more than one threshold value, for instance inorder to satisfy multiple (governmental) requirements. For example, athreshold value can be associated with the slope of a color value vs.time relation (i.e. the second derivative) and likewise multiplethreshold values can be associated with multiple slope values. Thisallows for measuring multiple sensitivities and/or multiple analytes.

In a third embodiment, a detection arrangement can be used comprising ofa processor, a memory, a display, and a color measuring device, saidmemory, said display and said color measuring device being arranged tocommunicate with said processor, by generating light signals with saidcolor measuring device, sending said light signals to said sample,receiving reflected light signals back from said sample, converting saidreflected light signals into color signals and sending said colorsignals to said processor. Optionally, said color signals aremanipulated in order to achieve a better separation between the colorcomponents of interest.

One example is adaptation of the color parameter of interest by means ofa mathematical formula such as for instance those present inphotographic manipulation programs known to the person skilled in theart. It has been shown that the use of such photographic manipulationprograms results in a marked difference in color parameters therebyallowing for easier discrimination between various samples. Thisphenomenon results in earlier interpretation of the assay in question,thereby making the assay significantly faster than prior art assays.

Another example of a mathematical formula is the calculation of thevalue of a composite parameter with the aid of said processor. Such aparameter may be a parameter Z in accordance with a following equation:

$Z = {\sum\limits_{i = 1}^{n}\; {w_{i}x_{i}}}$

where x_(i) is a color signal i and w_(i) is a corresponding weighingfactor and i is an index ranging from 1 to n and n is an integer equalto the number of color signals. In order to establish the ongoingchanges in the assay, the following sequence of steps may be performed:

-   -   1) measuring the value of Z for each sample and determining the        time t₁ at which said value Z is equal to a value Z₁ and the        time t₂ at which said value Z is equal to a value Z₂;    -   2) calculating by means of said processor the difference Δt        between said time t₁ and said time t₂ according to the formula        Δt=t₂−t₁        A suitable color model for use in the present invention is the        L*a*b model and said equation is:

Z=w ₁ ·L+w ₂ ·a+w ₃ ·b

Typical examples of suitable values of Z₁ and Z₂ are between 30 and −30provided that Z₁ is larger than or equal to Z₂. In addition, the abovesequence may be expanded by calculating if Δt is larger than Δt_(ref)and if this condition is met assigning a positive assay resultindicating that the concentration of said analyte is higher than aconcentration A and if this condition is not met, assigning a negativeassay result indicating that the concentration of said analyte is lowerthan a concentration B, wherein concentration A is smaller thanconcentration B.

In a most preferred example the detection arrangement of the presentinvention is used according to the scheme outlined in FIG. 4. Theprocess from generating light signals to obtaining assay data is givenin detail in the legend to FIG. 4.

In a fourth embodiment multiple samples may be employed, one of whichmay comprise a reference sample with a known amount (such as a thresholdamount) of the analyte to be detected. Thus, at least two samples ofdifferent fluids are contacted with an assay medium comprising an assaymicroorganism and an indicator, incubated and monitored simultaneouslyand wherein the value Z₁ is set by said processor to be equal to thelowest Z-value of any of the samples provided said lowest Z-value is atleast 10% and not more than 50% below the highest Z-value of any samplemeasured and wherein the value Z₂ is set by the processor to be equal tothe lowest Z-value of the samples provided said lowest Z-value is atleast 90% and not more than 200% below the highest Z-value of any samplemeasured.

In a fifth embodiment there is provided a method wherein Z₁ mentionedabove is equal to Z₂. and Z_(t) is the recorded value of Z at time t,and which method further comprises calculating by means of saidprocessor parameters of a mathematical expression relating Z_(t) to t,comparing the values of the parameters with those of a control sample inwhich the concentration of said analyte is known, and generating asignal indicating that the concentration of said analyte in said sampleis higher than or equal to or lower than the concentration of saidanalyte in the control sample. Optionally, the parameters of amathematical expression relating Z_(t) to t in said control sample arepre-set in a computer program and/or obtainable by said processor from aremote processor by means of a network.

In a sixth embodiment there is provided a method for determining thepresence or absence of an analyte in a fluid by analysis of image datafrom an assay that generates an image result on an assay medium. Saidimage results are obtained continuously at regular intervals and arecompared with stored reference data. These stored reference data consistof e.g. spectral changing curves of several known analytes at differentconcentrations. A comparison of the stored and obtained datasubsequently gives additional information concerning the type of analyteand the concentration of this analyte in the sample. It has surprisinglybeen found that the method of the present invention has the additionaladvantage that both various types of analytes and the concentration ofthe analytes in the sample can be detected simultaneously. Thus, bymeasuring spectral changing curves of known analytes and storing thesecurves in a processor, the specific assay results can be fitted to thestored data using a best-fit method and consequently the type andconcentration of analyte can be deduced. Using the approach of thisembodiment, a differentiation can be made between two or more differenttypes of analytes, such as for instance β-lactam antibiotics andsulfa's, like for instance between penicillin and sulfadiazin.

LEGEND TO THE FIGURES

FIG. 1 is an overview of an example of the detection and incubationarrangement of the present invention. A is a section comprising a colormeasuring device B, for instance a scanner, a means C for maintaining aconstant temperature or a temperature profile of the sample, such as aheating device comprising temperature sensors, and a communicationdevice D. E is a (personal) computer comprising a communication deviceD, an algorithm for determination of the Z-value F, one or moretemperature controllers G and software for interfacing with user(s)and/or external equipment H.

FIG. 2 is a detail of means C and G described in FIG. 1 comprising atemperature set-point generator 1, a controller (for instance PID) J, aheater element K, an assay bracket L and a temperature sensor M. Toincrease temperature accuracy means J up to and including M can be buildup several times.

FIG. 3 is a cross-sectional overview of an example of a heater deviceand assay bracket as described in FIG. 2 comprising a glass panel N, aheater O, a bracket P, an assay Q, a second heater R and insulationmaterial S.

FIG. 4 is an overview of the sequence of steps to be carried out usingthe detection and incubation arrangement of the present invention.

-   -   In T1, after each measurement the image from color measuring        device B is depicted as a set of pixels. Each pixel contains        three color signals: Red, Green and Blue (RGB). Pixel color can        be expressed as RGB but other color spaces can be used too, for        example Cyan, Magenta, Yellow, and Key (CMYK), XYZ and Hue,        Saturation and Value (HSV). The exact location of the assay is        determined using technology that is well-known to the person        skilled in the art, for instance such as described in WO        2003/034341. Next, a representative sample of an area of the        assay is taken and results in a matrix of n*m pixels (n and m        are preferably in the range of 1-1000, more preferably from        3-100, depending on the size of the assay; suitable examples of        n*m are 20*40, 100*100, 60*40, 20*20, most preferably 9*9).        Other shape possibilities to determine a representative sample        are circular, oval or a free set of pixels. The final result of        T1 is a set of pixels for each assay; each set contains RGB data        representing the color and luminance information of the selected        pixels.    -   In T2, all assay data are transferred into one representative        combination of RGB information. Examples of suitable methods are        calculating the mean or median.    -   In T3, depending on the optical measuring device and set-up,        color information may be calibrated. Already known calibration        methods, such as using color reference cards, can be applied. A        calibration cycle results in a calibration profile (Pr) that is        input to a correcting algorithm. In the current example an ICC        (intraclass correlation coefficient) profile is used. Other        calibrations methods may be used too. The result of T3 is a set        of corrected color signals RGB for the assay to be measured.    -   In T4 the RGB data is converted to the Lab color space using        known methods.    -   F is the algorithm mentioned under FIG. 1 for calculating the        Z-value: Z=w₁.L+w₂.a+w₃.b    -   T5 is an optional adaptation of the individual assay Z values        based on assay position and Z-value.    -   Optionally, in T6, for each assay successive samples may be        taken and smoothed, for example to exclude outliers and noise        reduction. Smoothing can be achieved by known methods such as        moving average or calculating the median from a number of        samples.    -   Optionally, in T7, in order to further reduce noise, a curve fit        may be applied onto consecutive samples. Examples of applicable        curves are polynomial, exponential and logarithmic types. A        preferred curve is a second degree polynomial curve.    -   Optionally, in T8, in order to compensate for possible        unfavorable assay effects, due for instance to storage        conditions, a normalizing step may be used. Initially the        compensation value Zs is 0, after m measurements the correction        value Zs can be calculated as follows:

$\sum\limits_{i = 1}^{60}\; {\sum\limits_{j = 1}^{96}\; {Z_{ij}/\left( {60*96} \right)}}$

-   -   -   wherein j is the number of assays, i the number of samples            over which the plate is normalized. Index i ranges from            1-120, preferably 10-90, most preferably 40-60 (the value 60            is given as an example in the formula).

    -   Function T9 is to determine when at least the required number of        assays (Nc) shows a negative result (Zr<Zth and Zth<Zcut-off).        As soon as this criterion is reached the results (positive or        negative) of each individual assay can be determined by T10.

    -   In T10, when indicated by T9 the final results Zu of each assay        Zr can be determined. The result is positive if Zr≧Zcut-off and        negative otherwise. When two different cut-off values are        applied, an additional result indicated as doubtful can be        obtained too.

    -   The conversion of RGB values to Lab and Z including further        processing to the final result Zu, is not limited to the        sequence as described above. In an alternative the RGB or Lab        values can be used directly to determine the final result.

FIG. 5 shows the differences in color profiles (expressed as Z-value,y-axis) for penicillin G (5A, left panel) and sulfadiazine (5B, rightpanel) as a function of assay duration (x-axis, in minutes). The curvesin FIG. 5A relate to samples containing, going from the lowest graph tothe highest graph, 0, 0.4, 0.6, 0.8, 1, 1.2, 1.6, 2, 2.4, 2.8 and 3.2ppb penicillin G. The curves in FIG. 5B relate to samples containing,going from the lowest graph to the highest graph, 0, 20, 40, 50, 60, 70,80, 100, 120, 140, 200, 300 and 400 ppb sulfadiazine. The followingequation was used: Z=0.35.a−0.65.b.

FIGS. 6-12 show the differences in color profiles (expressed as Z-value,y-axis) for seven antibiotics mentioned in the table below. The Z-valueis shown as a function of assay duration (x-axis, in minutes). Thecurves in these Figures relate to samples containing the antibiotics inincreasing concentrations (in ppb, see table). As outlined in thedetailed description, the following equation was used: Z=0.35.a−0.65.b.

Graph symbol (concentration in ppb) Figure number Antibiotic  ◯ X ♦ ▪ ▴6 Penicillin G 0 1 2 3 4 7 Sulfadiazin 0 25 50 100 150 200 8 Amoxicillin0 2 3 4 5 9 Ceftiofur 0 50 100 150 250 10 Cloxacillin 0 15 30 60 120 11Oxytetracyclin 0 100 200 300 400 12 Erythromycin 0 25 50 100 250

EXAMPLES Example 1 Determination of Color Values of in CommerciallyAvailable Microbial Inhibition Test for Two Different Antibiotics

Using the scanning technology outlined in the second aspect of theinvention, the color values in a commercially available microbialinhibition test (DSM DelvoTest®) were determined for a range ofconcentrations of penicillin G and sulfadiazin using the compositefunction Z=w_(L).L+w_(a).a+w_(b).b. Samples were kept at 64° C. by usinga titanium dioxide coated glass plate (obtained from Mansolar,www.mansolar.nl) equipped with a temperature sensor connected to aprocessor and two electrodes. The electrodes were connected to a 12 Vpower supply that was driven by the processor. The results of theseexperiments are shown in FIG. 5, showing that the curves for penicillinG and sulfadiazine differ substantially in shape. By relating theseshapes to curves stored in the memory of the processor, various analytes(here penicillin G vs. sulfadiazine) can be distinguished. Next to that,FIG. 5 also discloses, for instance for sulfadiazine, that a differencebetween a 0 ppb sample and a 20 ppb sample can be observed already after110 min, whereas prior art methods would have needed at least 150 minwhen an analyte-free sample reaches the Z=−5 to −10 region (where thecolor changes to yellow).

Example 2 Determination of Color Values of in Commercially AvailableMicrobial Inhibition Test Using Photographic Manipulation Programs

Using the scanning technology outlined in the second aspect of theinvention, the color values in a commercially available microbialinhibition test (DSM DelvoTest®), were determined for a range ofconcentrations of penicillin G, sulfadiazin and oxytetracyclin byanalysis using Adobe Elements as photographic manipulation program asfollows. The image obtained from the scanner was stored as .jpg file andopened in Adobe Elements. Using the darkpoint marker a positive test wasassigned and using the lightpoint marker a negative test was assigned.Subsequently the hue-value was adjusted to a value higher than 0(preferably 10-30) and the saturation was also adjusted to a valuehigher than 0 (preferably 100). Next the color values were determinedusing the formula Z=0.35.a−0.65.b. From the table below it becomes clearthat, using the photographic manipulation program, assay duration of 45min with sensitivities of 3 ppb penicillin G, 200 ppb sulfadiazin and200 ppb oxytetracyclin could be achieved.

Using photographic Assay dura- Penicillin Sulfa- Oxytetracy-manipulation tion (min) G (ppb) diazin (ppb) clin (ppb) No52 >6 >300 >500 Yes 45 3 200 200

Example 3 Determination of Color Values of in Commercially AvailableMicrobial Inhibition Test for Two Different Antibiotics

Using the scanning technology (shown in FIGS. 1-3) and outlined in thesecond aspect of the invention, the color values in a commerciallyavailable microbial inhibition test DSM DelvoTest®, were determined fora range of concentrations of penicillin G, sulfadiazin, ceftiofur,amoxicillin, cloxacillin, oxytetracyclin and erythromycin using thecomposite function Z=w_(L).L+w_(a).a+w_(b).b.

Sensitivity Sensitivity Example 3 DelvoTest ® SP-NT Antibiotic (in ppb,after 105 min) (in ppb, after 150 min) Penicillin G 2 1-2 Sulfadiazin 5025-50 Amoxicillin 3 2-3 Ceftiofur 100 25-50 Cloxacillin 15 20Oxytetracyclin 200 250-500 Erythromycin 100 40-80Samples were kept at 64° C. using a plate with holes. Heat traces aredivided over the plate, which allows generating a specific heat inputrelated to the position, for homogeneous temperature gradients. Toincrease accuracy of the temperature gradient over the plate,temperature sensors and controllers were used to adjust the currentinput. Temperature was controlled by means of a PID. To increase speedof detection and reproducibility the heating device was pre-heated at atemperature of 80° C. The results of these experiments are shown inFIGS. 6-12 and the table above, showing that for said antibiotics therequired concentration detection limits are obtainable within 105minutes, whereas prior art methods, such as DelvoTest® SP-NT would haveneeded at least 150 min, when an analyte-free sample reaches the Z=−5 to−12 region (where the color changes to yellow).

1. Detection arrangement for detecting presence of an analyte in asample, comprising a processor, a memory, a display, and a colormeasuring device, said memory, said display and said color measuringdevice being arranged to communicate with said processor, said colormeasuring device being arranged to generate light signals, to send saidlight signals to said sample, to receive return light signals from saidsample, to convert said return light signals into color signals and tosend said color signals to said processor, said processor being operatedby instructions stored in said memory and being arranged to calculate avalue of a composite parameter Z in accordance with a followingequation: $Z = {\sum\limits_{i = 1}^{n}\; {w_{i}x_{i}}}$ where x, isa color signal i and w, is a corresponding weighing factor and i is anindex ranging from 1 to n and n is an integer equal to the number ofcolor signals, characterized in that a means is present for maintaininga constant temperature or a temperature profile of said sample. 2.Detection arrangement according to claim 1 wherein said means formaintaining a constant temperature or a temperature profile is atransparent plate coated with titanium dioxide to which electrodes areattached.
 3. Detection arrangement according to claim 1 wherein saidmeans for maintaining a constant temperature or a temperature profile isa device made from metal comprising apertures.
 4. A method fordetermining the presence or absence of an analyte in a fluid by analysisof image data from an assay that generates an image result on an assaymedium, comprising the steps of: (a) incubating a sample of said fluidtogether with said assay at a pre-set temperature or temperature profile(b) obtaining said image result on an assay medium; and (c) imaging theimage result with an image acquisition device to generate digital imagedata corresponding to the image result; and (d) using data processingmeans, applying to the digital image data a stored relationship betweenthe image result and assay calibration data to generate a quantifiedresult for said assay, characterized in that incubation step (a) iscarried out simultaneously with steps (b)-(c).
 5. A method according toclaim 4 wherein multiple image results are obtained continuously or atregular intervals in time.
 6. A method according to claim 4 furthercomprising photographic manipulation.
 7. A method according to claim 4wherein the following steps are performed: (a) measuring the value of Zfor each sample and determining the time t₁ at which said value Z isequal to a value Z₁ and the time t₂ at which said value Z is equal to avalue Z₂; (b) calculating by means of said processor the difference Δtbetween said time t₁ and said time t₂ according to the formula Δt=t₂−t₁8. A method according to claim 4 wherein said quantified result isgenerated in less than 120 minutes.